Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Intracellular Expression of CTB in Vibrio cholerae Strains in Laboratory Culture Conditions

  • Hunseok Choi (Department of Pharmacy, College of Pharmacy, Hanyang University) ;
  • Seonghyeon Son (Department of Pharmacy, College of Pharmacy, Hanyang University) ;
  • Donghyun Lee (Department of Pharmacy, College of Pharmacy, Hanyang University) ;
  • Jonghyun Bae (Department of Pharmacy, College of Pharmacy, Hanyang University) ;
  • Eunyoung Seo (Department of Pharmacy, College of Pharmacy, Hanyang University) ;
  • Dong Wook Kim (Department of Pharmacy, College of Pharmacy, Hanyang University) ;
  • Eun Jin Kim (Department of Pharmacy, College of Pharmacy, Hanyang University)
  • Received : 2023.02.09
  • Accepted : 2023.03.29
  • Published : 2023.06.28
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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 7th to the 20th 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

Acknowledgement

This work was supported by grants NRF-2021R1A2C1010857 and NRF-RS-2023-00208573 from the National Research Foundation (NRF) of Korea.

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