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Open Access Research article

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

Yukihiro Hiramatsu1, Masatatsu Yamamoto2, Tomomitsu Satho1*, Keiichi Irie1, Akiko Kai1, Saori Uyeda1, Yuki Fukumitsu1, Akihisa Toda3, Takeshi Miyata4, Fumio Miake1, Takeshi Arakawa56 and Nobuhiro Kashige1

Author Affiliations

1 Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka, Japan

2 Department of Molecular Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1, Sakuragaoka, Kagoshima, Japan

3 Daiichi College of Pharmaceutical Sciences, 22-1, Tamagawa-cho, Minami-ku, Fukuoka, Japan

4 Division of Molecular Functions of Food, Department of Biochemistry and Biotechnology, Kagoshima University, 1-21-24, Korimoto, Kagoshima, Japan

5 Molecular Microbiology Group, Department of Tropical Infectious Diseases, COMB, Tropical Biosphere Research Center, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, Japan

6 Division of Host Defense and Vaccinology, Department of Microbiology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa, Japan

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BMC Biotechnology 2014, 14:38  doi:10.1186/1472-6750-14-38

Published: 10 May 2014

Abstract

Background

Lactobacillus species are used as bacterial vectors to deliver functional peptides to the intestine because they are delivered live to the intestine, colonize the mucosal surface, and continue to produce the desired protein. Previously, we generated a recombinant Lactobacillus casei secreting the cholera toxin B subunit (CTB), which can translocate into intestinal epithelial cells (IECs) through GM1 ganglioside. Recombinant fusion proteins of CTB with functional peptides have been used as carriers for the delivery of these peptides to IECs because of the high cell permeation capacity of recombinant CTB (rCTB). However, there have been no reports of rCTB fused with peptides expressed or secreted by Lactobacillus species. In this study, we constructed L. casei secreting a recombinant fusion protein of CTB with YVAD (rCTB–YVAD). YVAD is a tetrapeptide (tyrosine–valine–alanine–aspartic acid) that specifically inhibits caspase-1, which catalyzes the production of interleukin (IL)-1β, an inflammatory cytokine, from its inactive precursor. Here, we examined whether rCTB–YVAD secreted by L. casei binds to GM1 ganglioside and inhibits caspase-1 activation in Caco-2 cells used as a model of IECs.

Results

We constructed the rCTB–YVAD secretion vector pSCTB–YVAD by modifying the rCTB secretion vector pSCTB. L. casei secreting rCTB–YVAD was generated by transformation with pSCTB–YVAD. Both the culture supernatant of pSCTB–YVAD-transformed L. casei and purified rCTB–YVAD bound to GM1 ganglioside, as did the culture supernatant of pSCTB-transformed L. casei and purified rCTB. Interestingly, although both purified rCTB–YVAD and rCTB translocated into Caco-2 cells, regardless of lipopolysaccharide (LPS), only purified rCTB–YVAD but not rCTB inhibited LPS-induced caspase-1 activation and subsequent IL-1β secretion in Caco-2 cells, without affecting cell viability.

Conclusions

The rCTB protein fused to a functional peptide secreted by L. casei can bind to GM1 ganglioside, like rCTB, and recombinant YVAD secreted by L. casei may exert anti-inflammatory effects in the intestine. Therefore, rCTB secreted by L. casei has potential utility as a vector for the delivery of YVAD to IECs.

Keywords:
Caspase-1; Cholera toxin B subunit; GM1 ganglioside; Interleukin-1β; Lactobacillus casei; YVAD