Intracellular Expression of CTB in Vibrio cholerae Strains in Laboratory Culture Conditions

Abstract

The introduction of the toxT-139F allele triggers the expression of TCP (toxin co-regulated pilus) and CT (cholera toxin) under simple laboratory culture conditions in most Vibrio cholerae strains. Such V. cholerae strains, especially strains that have been used in OCVs (oral cholera vaccines), can induce antibody responses against TCP in animal models. However, CT produced in these V. cholerae strains is secreted into the culture medium. In this study, V. cholerae strains that can express intracellular CTB under the control of the toxT-139F allele have been constructed for potential application in OCVs. First, we constructed a recombinant plasmid directly linking the ctxAB promoter to ctxB without ctxA and confirmed CTB expression from the plasmid in V. cholerae containing the toxT-139F allele. We constructed another recombinant plasmid to express NtrCTB, from which 14 internal amino acids-from the 7^th to the 20^th amino acid-of the leader peptide of CTB have been omitted, and we found that NtrCTB remained in the cells. Based on those results, we constructed V. cholerae strains in which chromosomal ctxAB is replaced by ntrctxB or ntrctxB-dimer. Both NtrCTB and NtrCTB-dimer remained in the bacterial cells, and 60% of the NtrCTB-dimer in the bacterial cells was maintained in a soluble form. To develop improved OCVs, these strains could be tested to see whether they induce immune responses against CTB in animal models.

Keywords: Cholera; Vibrio cholerae; cholera toxin (CT); cholera toxin B subunit (CTB); vaccine.

Fig. 1. Secretion of cholera toxin in V. cholerae strains.

The whole culture (culture), culture supernatant (sup.), and cell fractions (cell) were prepared as described in the Methods and analyzed by Western blotting using anti-CT. The CT expression of two V. cholerae strains, YJB001 and YJB020, which are toxT-139F derivatives of the classical biotype strain O395 and the Wave 3 El Tor biotype strain IB5230, respectively, was analyzed. CTA, CTA1, and CTB are indicated by black, white, and shaded arrows, respectively.

Fig. 2. Expression of CTB from a recombinant plasmid that contains the ctxB ORF linked to the ctxAB promoter.

(A) Expression of CTB in YJB020 (lane 1), DH5α-pUC18-ctxB (lane 2), EJK002-pUC18-ctxB cultured in AKI broth (lane 3), EJK002-pUC18-ctxB cultured in LB medium (lane 4), EJK001-pUC18-ctxB cultured in AKI broth (lane 5), and EJK001-pUC18-ctxB cultured in LB medium (lane 6). Bacteria were cultured at 37°C. (B) Secretion of CTB from V. cholerae strains that express CTB from pUC18-ctxB (EJK002-pUC18-ctxB). Culture (lanes 1 and 4), culture supernatant (lanes 2 and 5), and cells (lanes 3 and 6) of YJB020 and EJK002-pUC18-ctxB were analyzed. Bacteria were cultured in AKI broth at 37°C.

Fig. 3. Expression of CTB variants in V. cholerae strains that harbor pUC18-ctxB, pUC18-ntrctxB, or pUC18- ntrctxB-dimer.

(A) Expression of CTB and NtrCTB in V. cholerae strains harboring pUC18-ctxB and pUC18-ntrctxB, respectively. Culture (lanes 1, 4, and 7), culture supernatant (lanes 2, 5, and 8), and cells (lanes 3, 6, and 9) of YJB020 (lanes 1–3), EJK002-pUC18-ctxB (lanes 4–6), and EJK002-pUC18-ntrctxB (lanes 7, 8, and 9) that were cultured in AKI broth at 37°C were analyzed by Western blotting using anti-CT. CTB and NtrCTB are indicated by white and black arrows, respectively. (B) Expression of CTB and NtrCTB-dimer in V. cholerae strains that harbor pUC18-ctxB-dimer. Culture (lanes 1 and 4), culture supernatant (lanes 2 and 5), and cells (lanes 3 and 6) of YJB020 (lanes 1–3) and EJK002-pUC18-ntrctxB-dimer (lanes 4–6) that were cultured in AKI broth at 37°C were analyzed. CTB and NtrCTB-dimer are indicated by white and black arrows, respectively.

Fig. 4. Expression of CTB variants in V. cholerae strains in which ctxB, ntrctxB, and ntrctxB-dimer were integrated into the chromosome.

The expression of variant CTB was analyzed by Western blotting with anti-CT (A) and anti-CTB (B and C). (A) EYS003 expressing CTB, (B) HSC001 expressing NtrCTB, and (C) HSC002 expressing NtrCTB-dimer. M: molecular weight marker, lane 1: culture of YJB020, lane 2: culture, lane 3: culture supernatant, and lane 4: cells of each strain.

Fig. 5. CTB dimer expressed in V. cholerae strain remains in soluble fractions.

YJB020 and HSC002 were cultured in AKI broth at 37°C. Then, the whole culture of YJB020 (lane 1), harvested cells of HSC002 (lane 2), soluble fraction (lane 3), and insoluble fraction (lane 4) of HSC002 were analyzed by Western blotting using anti-CTB.