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Molecular characterization of the fragilysin pathogenicity islet of enterotoxigenic Bacteroides fragilis
J S Moncrief1, A J Duncan, R L Wright
1Department of Biochemistry, Fralin Center for Biotechnology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0346, USA. jmoncrie@vt.edu
Abstract:
Enterotoxigenic strains of Bacteroides fragilis produce an extracellular metalloprotease toxin (termed fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the fragilysin gene is contained within a small genetic element termed the fragilysin pathogenicity islet. The pathogenicity islet of B. fragilis VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the B. fragilis chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.
Insights
Enterotoxigenic Bacteroides fragilis harbors the fragilysin gene on a mobile genetic element called the pathogenicity islet. This islet integrates into specific chromosomal sites, potentially influencing toxin production and bacterial virulence.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Enterotoxigenic Bacteroides fragilis produces fragilysin, a metalloprotease toxin.
- Fragilysin causes intestinal epithelial cell damage, fluid secretion, and tissue injury.
Purpose of the Study:
- To characterize the genetic element containing the fragilysin gene.
- To investigate the integration mechanism of this genetic element in the Bacteroides fragilis chromosome.
Main Methods:
- DNA sequencing of the fragilysin pathogenicity islet.
- Polymerase Chain Reaction (PCR) analysis of multiple bacterial strains.
- Chromosomal integration site analysis.
Main Results:
- The fragilysin gene is located on a 6,033 bp genetic element, the fragilysin pathogenicity islet, flanked by direct repeats.
- The islet integrates at a specific chromosomal locus in enterotoxigenic strains.
- A distinct GC-rich sequence was identified at the integration site in non-toxigenic strains, suggesting it as a potential integration target.
- An additional open reading frame encoding a putative protein similar to fragilysin was identified within the islet.
Conclusions:
- The fragilysin pathogenicity islet is a mobile genetic element responsible for carrying the fragilysin gene.
- Chromosomal integration of the islet occurs at a specific site, with non-toxigenic strains lacking a characteristic target sequence.
- The presence of a second, similar gene suggests potential functional redundancy or evolution of the toxin system.