Deletion of peb4 gene impairs cell adhesion and biofilm formation in Campylobacter jejuni
Hiroshi Asakura
Division of Biomedical Food Research, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan
Search for more papers by this authorManabu Yamasaki
Microbial Chemistry Research Foundation, Sinagawa-ku, Tokyo, Japan
Search for more papers by this authorShigeki Yamamoto
Division of Biomedical Food Research, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan
Search for more papers by this authorShizunobu Igimi
Division of Biomedical Food Research, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan
Search for more papers by this authorHiroshi Asakura
Division of Biomedical Food Research, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan
Search for more papers by this authorManabu Yamasaki
Microbial Chemistry Research Foundation, Sinagawa-ku, Tokyo, Japan
Search for more papers by this authorShigeki Yamamoto
Division of Biomedical Food Research, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan
Search for more papers by this authorShizunobu Igimi
Division of Biomedical Food Research, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan
Search for more papers by this authorEditor: Stephen Smith
Abstract
Campylobacter jejuni is a microaerophilic bacterium that causes diarrhea in humans. The first step in establishing an infection is adherence to a host cell, which involves two major cell-binding proteins, Peb1A (CBF1) and Peb4 (CBF2). Because the functional role of Peb4 on the cell adhesion remains unclear compared with that of Peb1A, a C. jejuni peb4 deletion mutant was constructed and cell adherence and ability to colonize mouse intestine were studied. The result showed that adherence of the peb4 mutant strain to INT407 cells was 1–2% that of the wild-type strain. Mouse challenge experiments showed a reduced level and duration of intestinal colonization by the mutant compared with the wild-type strain. In addition, fewer peb4 mutant cells than wild-type cells responded to stress by forming a biofilm. Proteomic analysis revealed that the expression levels of proteins involved in various adhesion, transport, and motility functions, which are required for biofilm formation by the pathogen, were lower in the peb4 mutant than in the wild-type strain. A Peb4 homolog has prolyl cis/trans-isomerase activity, suggesting that the loss of this activity in the mutant strain may be responsible for the repression of these proteins.
Supporting Information
Fig. S1. Abundance of specific RNAs assessed by real-time RT-PCR analysis of C. jejuniwild-type and peb4 mutant strains. (A) peb1A mRNA, (B) peb4 mRNA, (C) flaA mRNA, (D) flaB mRNA, (E) luxS mRNA. Bacteria was grown in 1 ml of MHB at 37????C under microaerobic condition, and total RNA was prepared with RiboPure bacteria kit (Ambion), and reverse-transcribed to synthesize cDNA with the Transcriptor 1st strand cDNA synthesis kit (Roche). The target genes were detected with SYBR Green I (Roche) with primer pairs as follows: peb1A (5' - TTTAGCTAG CAATTTGGCAACATC -3' and 5' - CCGCATTATGCTT TACTTGATCAA-3' ), peb4 (5' - GGTGAGCTTGGTTGGTTTGATC-3' and 5' - AAGCGCGAAAGCAGC ATCT-3' ), flaA (5' - CGGCCATAGTTTTCTTTCA TATCAG- 3' and 5' -AAGGTAGAGGGATTAAAATCGATGGT- 3' ), flab (5' -AAACATTTTGCTGCACTGCAT-3' and 5' -ATATAACATCCTTGCGCAA TCAG-3, ), luxS (5' - TAAGCGATCAAAGCAAAATTCCT-3' and 5' -TCTAA AGAATGCATTGCGCAAGT-3' ), and 16s rRNA (5' -AGCAGCCGCGGTAA TACG-3' and 5' -GCCCTTTACGCCCAGTGATT-3' ). Gene expression analysis was performed by relative quantification with 16s rRNA as the reference gene. Normalized ratio was calculated as follows: (concentration (conc.) of target/ conc. of reference) sample vs (conc. of target/ conc. of reference) calibrator. Table S1. Summary of C. jejuni proteins modulated either between strains 11168 and peb4 mutant , or between planktonic and biofilm cells.
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