Abstract
Vibrio cholerae is a Gram-negative, facultative anaerobic bacterial species that causes serious disease and can grow on various carbon sources, including chitin polysaccharides. In saltwater, its attachment to chitin surfaces not only serves as the initial step of nutrient recruitment but is also a crucial mechanism underlying cholera epidemics. In this study, we report the first characterization of a chitooligosaccharide-specific chitoporin, VcChiP, from the cell envelope of the V. cholerae type strain O1. We modeled the structure of VcChiP, revealing a trimeric cylinder that forms single channels in phospholipid bilayers. The membrane-reconstituted VcChiP channel was highly dynamic and voltage induced. Substate openings O1', O2', and O3', between the fully open states O1, O2, and O3, were polarity selective, with nonohmic conductance profiles. Results of liposome-swelling assays suggested that VcChiP can transport monosaccharides, as well as chitooligosaccharides, but not other oligosaccharides. Of note, an outer-membrane porin (omp)-deficient strain of Escherichia coli expressing heterologous VcChiP could grow on M9 minimal medium supplemented with small chitooligosaccharides. These results support a crucial role of chitoporin in the adaptive survival of bacteria on chitinous nutrients. Our findings also suggest a promising means of vaccine development based on surface-exposed outer-membrane proteins and the design of novel anticholera agents based on chitooligosaccharide-mimicking analogs.
Highlights
Vibrio cholerae, a Gram-negative bacterium of the genus Vibrio, is a human pathogen causing the life-threatening waterborne disease cholera
Further microarray expression experiments identified an ORF in mRNAs of V. cholerae expressed in response to growth of the bacteria on chitin oligosaccharides, and VC0972 was identified as the chip gene encoding chitoporin (ChiP), which serves as a chitooligosaccharide transporter [12]
The chip fragment was cloned into the pET23d [1] expression plasmid, and the recombinant VcChiP was expressed in the outer membrane protein-deficient Escherichia coli BL21(Omp8) strain
Summary
A Gram-negative bacterium of the genus Vibrio, is a human pathogen causing the life-threatening waterborne disease cholera. OmpU (38 kDa) and OmpT (40 kDa) were shown to be voltage-sensitive and cation-selective [14] Both OmpT and OmpU were later shown to allow the passage of charged amino acids and antibiotic molecules [15]. Another V. cholerae porin, identified as VCA1008, was shown to be selective for anions, such as phosphate [16]. Further microarray expression experiments identified an ORF in mRNAs of V. cholerae expressed in response to growth of the bacteria on chitin oligosaccharides, and VC0972 was identified as the chip gene encoding chitoporin (ChiP), which serves as a chitooligosaccharide transporter [12]. Cell growth experiments were carried out to verify the important role of VcChiP in the growth of the bacteria on chitooligosaccharides
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