Salmonella enteritidis FliC (Flagella Filament Protein) Induces Human β-Defensin-2 mRNA Production by Caco-2 Cells

Ken-Ichi Ogushi,Akihiro Wada,Takuro Niidome,Naoki Mori,Kazunori Oishi,Tsuyoshi Nagatake,Akira Takahashi,Hiroshi Asakura,Sou-Ichi Makino,Hironobu Hojo,Yoshiaki Nakahara,Mio Ohsaki,Tomomitsu Hatakeyama,Haruhiko Aoyagi,Hisao Kurazono,Joel Moss,Toshiya Hirayama

doi:10.1074/jbc.m011618200

Abstract

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a approximately 2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-kappaB (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-kappaB activation and thus plays an important role in up-regulation of the innate immune response.

Highlights

Antimicrobial peptides are crucial for host defense at mucosal surfaces
After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis
Bacteria Strains and Growth Conditions—S. enteritidis, S. typhimurium, S. typhi, and S. dublin strains were grown on tryptic soy agar and in liquid culture overnight in 3 ml of tryptic soy broth (TSB)

Summary

Introduction

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human ␤-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. The hBD-2-inducing protein in S. enteritidis supernatant was purified by sequential anion exchange and gel filtration chromatography, and the amino-terminal sequence was determined. It was established that the hBD-2-inducing factor is flagella filament structural protein (FliC) of S. enteritidis, and induction results from NF-␬B activation

Methods

Results

Conclusion