title: 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.
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.
authors.forename: Yukihiro
authors.initials: Y
authors.lastname: Hiramatsu
authors.forename: Masatatsu
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1, Nanakuma, Jonan-ku, Fukuoka, Japan. satho@cis.fukuoka-u.ac.jp.
authors.forename: Tomomitsu
authors.initials: T
authors.lastname: Satho
authors.forename: Keiichi
authors.initials: K
authors.lastname: Irie
authors.forename: Akiko
authors.initials: A
authors.lastname: Kai
authors.forename: Saori
authors.initials: S
authors.lastname: Uyeda
authors.forename: Yuki
authors.initials: Y
authors.lastname: Fukumitsu
authors.forename: Akihisa
authors.initials: A
authors.lastname: Toda
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyata
authors.forename: Fumio
authors.initials: F
authors.lastname: Miake
authors.forename: Takeshi
authors.initials: T
authors.lastname: Arakawa
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Kashige
mesh_terms: D000595:Amino Acid Sequence; D018938:Caco-2 Cells; D020170:Caspase 1 / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism*; D007780:Lacticaseibacillus casei / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000633:toxicity; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology
chemical_list: D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; D020170:Caspase 1